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Aneuploid progeny of the American oyster, Crassostrea

virginica, produced by tetraploid x diploid crosses: another example of chromosome instability in polyploid oysters

Journal: Genome

Manuscript ID gen-2015-0222.R1

Manuscript Type: Article

Date Submitted by the Author: 08-Feb-2016

Complete List of Authors: Sousa, Joana; Virginia Institute of Marine Science, Fisheries

Allen, Standish; Virginia Institute of Marine Sciences, Baker, Haley; The University of Alabama Matt, Joseph; Virginia Institute of Marine Science, Fisheries

Keyword: Crassostrea virginica, aneuploidy, triploidy, mitotic instability, chromosomes

https://mc06.manuscriptcentral.com/genome-pubs

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Aneuploid progeny of the American oyster, Crassostrea virginica, produced by tetraploid x

diploid crosses: another example of chromosome instability in polyploid oysters.

Authors’ names:

Joana Teixeira de Sousa1*, Standish K. Allen, Jr.

1, Haley Baker

2, Joseph L. Matt

1

Addresses:

1 Aquaculture Genetics and Breeding Technology Center. Virginia Institute of Marine

Science. Gloucester Point, VA 23062, USA.

2 The University of Alabama. Tuscaloosa, AL 35487, USA.

* Corresponding author

Phone: 804.684.7896 - Fax: 804.684.7717

Email: [email protected]

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Abstract

The commercial production of triploids, and the creation of tetraploid broodstock to support

it, has become an important technique in aquaculture of the eastern oyster, Crassostrea virginica.

Tetraploids are produced by cytogenetic manipulation of embryos and have been shown to

undergo chromosome loss (to become a mosaic) with unknown consequences for breeding. Our

objective was to determine the extent of aneuploidy in triploid progeny produced from both

mosaic and non-mosaic tetraploids. Six families of triploids were produced using a single

diploid female and crossed with three mosaic and non-mosaic tetraploid male oysters. A second

set of crosses was performed with the reciprocals. Chromosome counts of the resultant embryos

were tallied at 2-4 cell stage and as 6-hour(h)-old embryos. A significant level of aneuploidy

was observed in 6-h-old embryos. For crosses using tetraploid males, aneuploidy ranged from

53 – 77% of observed metaphases, compared to 36% in the diploid control. For crosses using

tetraploid females, 51 – 71% of metaphases were aneuploidy versus 53% in the diploid control.

We conclude that somatic chromosome loss may be a regular feature of early development in

triploids, and perhaps polyploid oysters in general. Other aspects of chromosome loss in

polyploid oysters are also discussed.

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Key words: Crassostrea virginica, aneuploidy, triploidy, mitotic instability, chromosomes

Résumé

La production commerciale d’huîtres triploïdes, ainsi que la création de stocks de géniteurs

tétraploïdes pour la soutenir, sont devenues des techniques importantes dans l'aquaculture de

l'huître américaine, Crassostrea virginica. Les tétraploïdes sont produites grâce à une

manipulation cytogénétique des embryons, qui peut provoquer la perte de chromosomes

(devenant mosaïque) présentant des conséquences inconnues pour la reproduction. L’objectif de

cette étude était de déterminer l’impact de l’aneuploïdie chez les descendants triploïdes produits

à partir de tétraploïdes aussi bien mosaïques que non-mosaïques. Six familles de triploïdes ont

été produites en utilisant une femelle diploïde croisée avec trois mâles tétraploïdes mosaïques et

non-mosaïques. Une deuxième série a été réalisée en utilisant des croisements réciproques. Le

nombre de chromosomes ont été comptés sur les embryons au stade « 2-4 cellules » et 6 heures

après la fécondation. Après 6 heures, un niveau significatif d’aneuploïdie a été observé. Pour les

croisements impliquant les mâles tétraploïdes, dans les métaphases observées, le niveau

d’aneuploïdie était compris entre 53 et 57% contre 36% dans le contrôle diploïde. Pour les

croisements impliquant les femelles tétraploïdes, le niveau d’aneuploïdie était compris entre 51

et 71% contre 53% dans le contrôle diploïde.

Nous pouvons conclure que la perte de chromosomes somatiques pourrait être une

caractéristique normale observée lors du développement précoce chez les triploïdes, et de façon

plus générale chez les huîtres polyploïdes. D'autres aspects liés à la perte de chromosomes chez

les huîtres polyploïdes sont également abordés au cours de cette étude.

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Mots-clés: aneuploïdie, triploïdie, l'instabilité mitotique, chromosomes

Introduction

The eastern oyster, Crassostrea virginica, is a highly fecund commercial bivalve and well-

adapted to an estuarine existence, being highly tolerant to wide fluctuations of temperature,

salinity, suspended sediments, and dissolved oxygen (Kennedy et al. 1996). However, the

eastern oyster has declined in many estuaries where it was once abundant due to over-harvesting,

habitat degradation, reduced water quality, disease, and interactions among these factors

(Kingsley-Smith 2009). In order to overcome the reduction in commercial product, oyster

aquaculture has become prominent in the last decade led by genetic improvements, such as

selection (Guo 2009, Frank-Lawale et al. 2014) and polyploidy (Dégremont et al. 2012).

Polyploidy is the heritable condition of possessing an additional set (or sets) of

chromosomes, being a phenomenon well tolerated in many groups of eukaryotes such as plants,

fish, and amphibians (Comai 2005). In bivalves, natural polyploidy is less common but has been

observed in two marine species of genera Lasaea: Lasaea australis (Foighil and Thiriot-

Quievreux 1991) and Lasaea consanguinea (Thiriot-Quiévreux et al. 1988), in the freshwater

clams Sphaerium striatinum (Lee 1999) and Sphaerium rhomboideum (Petkevičiūtė et al. 2007)

and in Corbicula spp. (Park et al. 2000). As evidenced from the high incidence of polyploidy in

some taxa, polyploids can clearly be advantageous. Such advantages can be obtained from

inducing polyploidy as well. For example, the commercial production of triploid oysters (Guo et

al. 1996) and the creation of tetraploids to serve as progenitors of hatchery-bred triploid spat

(Guo and Allen 1994a), has become an essential and successful tool in aquaculture (Nell 2002,

Piferrer et al. 2009, Guo et al. 2009).

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The first viable tetraploid oysters (Crassostrea gigas) were produced by treating fertilized

eggs of a triploid with cytochalasin B to block the release of the first polar body to create de

novo tetraploids (Guo and Allen 1994a). De novo tetraploids are genetically unique because they

are obtained from a triploid x diploid cross, so consequently the tetraploid genome is made up of

three chromosome sets from the mother (triploid) and one from the father (diploid). Moreover,

by definition, these types of tetraploids derive from “fertile” triploids (Guo and Allen 1994b,

Eudeline et al. 2000). Alternative methods to produce tetraploids exist (McCombie et al. 2005b,

Benabdelmouna and Ledu 2015), but for all types, subsequent generations of tetraploids are

propagated from tetraploid x tetraploid crosses (Guo and Allen 1997). Virtually, all commercial

triploid oysters in the world are now created using the tetraploid x diploid cross. Triploid oysters

have several advantages for oyster culture, especially reduced fecundity with consequent higher

growth and improved market quality during the reproductive season (Allen 1988).

Although polyploidy can be advantageous, it is often associated with cytological problems.

For example, polyploidy increases the occurrence of spindle irregularities, e.g., multiple spindles

that can lead to the chaotic segregation of chromatids and the production of aneuploid cells with

abnormal numbers of chromosomes (Borel et al. 2002). Across taxa, chromosome loss in

polyploids is frequent and occurs to a much greater extent than in diploids (Comai 2005). For

bivalves, cytogenetic abnormalities, such as aneuploidy, are common in diploid populations

(Thiriot-Quiévreux et al. 1992; Leitão et al. 2001; de Sousa et al. 2011) and this phenomenon is

apparent in de novo polyploid shellfish as well (Guo and Allen 1994a; Wang et al. 1999; Yang et

al. 1999; Yang et al. 2000). For example, induced triploid and tetraploid embryos of the scallop

Chlamys farreri had up to 53% of aneuploid (hypoploid) cells (Yang et al. 1999; Yang et al.

2000). High levels of aneuploidy also occurred in polyploid Pacific oysters, C. gigas, in both

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adults (Wang et al. 1999; McCombie et al. 2005a; Zhang et al. 2010a; Zhang et al. 2013) and

larvae (Guo and Allen 1994a; Guo and Allen 1997).

For oysters, chromosome loss is not limited to aneuploidy, but also includes the loss of entire

sets of chromosomes to become heteroploid mosaics (herein called “mosaics”) through a process

called reversion (Allen et al. 1996; Zhang et al. 2010a). It seems that cells undergoing reversion,

at least some of them, continuously eliminate chromosomes until a stable euploid state is

established (Zhang et al. 2013). The loss of chromosomes from tetraploids is of major scientific

interest and of practical concern for commercial oyster culture (Matt and Allen 2014). Previous

studies in C. virginica revealed that tetraploid mosaics have little impact on triploid production,

although this question was not examined at the chromosomal level (Matt and Allen 2014). Until

now, flow cytometry (FCM) was our principal research tool for detecting reversion. FCM data

can be rapidly obtained enabling high throughput, however, it is difficult to detect small

differences in DNA content and, consequently, the data contain little information about

aneuploidy.

We initiated this study with the intention of refining the information in Matt and Allen

(2014) with chromosome counts. To that end, we established crosses between mosaic and non-

mosaic tetraploids with reference diploids. In the course of the investigation, we uncovered a

surprisingly and unexpectedly high incidence of aneuploidy in early (6-hour[h]-old) embryos.

We then broadened our question by comparing these data to 2-4 cell embryos from the same

crosses to try to identify the source of aneuploidy.

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Materials and methods

Experimental population

Tetraploid C. virginica broodstock were obtained from lines propagated by the Aquaculture

Genetics and Breeding Technology Center (ABC). Tetraploid oysters were opened and males

and females sorted. From each tetraploid, a 4mm2 gill sample was dissected from one lamella

and processed for FCM (Allen et al. 1996). Gill cells were stained in DAPI/DMSO (Allen and

Bushek 1992) and analyzed on a Partec Cyflow Space flow cytometer. Samples were measured

with referencing to a diploid standard for mean relative DNA content as well as variation (CV) in

DNA content of cell populations. Gill samples were analyzed for somatic ploidy to obtain

tetraploids that had only tetraploid cells apparent (herein called “non-mosaics”) and to obtain

tetraploids that had multiple ploidy types in the somatic tissue (“mosaics”). The number of

mosaics were more numerous than non-mosaics (data not shown). Diploid gametes were

obtained from a single male or female, depending on the test crosses.

Crosses

After confirmation of ploidy in parents, males and females were strip spawned using the

technique outlined by Allen and Bushek (1992). In the first set of crosses, three tetraploid

mosaic males and three tetraploid non-mosaic males were crossed with a single diploid female

tester creating six half-sib groups. A control cross was made using the diploid female tester and

single male diploid. In a second set of reciprocal crosses, three tetraploid mosaic females and

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three tetraploid non-mosaic females were crossed to a single diploid male tester, producing

another set of six half sib groups. A control cross was made using the diploid male tester and

single female diploid. Thus, each set of reciprocal crosses comprised three mosaic tetraploids

and three non-mosaic tetraploids all crossed with the same diploid, and a control.

Cytogenetics

To block the mitosis in metaphase cells, about 300,000 1-h-old (2-4 cell embryos) and

300,000 6-h-old embryos were collected and incubated for 20 minutes in seawater containing

0.005% colchicine. The 1-h-old embryos were fixed directly in Carnoy’s solution-freshly

prepared absolute ethanol: acetic acid (3:1) (Guo and Allen 1997). The 6-h-old embryos were

treated for 10 minutes in 0.9% sodium citrate and then fixed in Carnoy’s. The fixed embryos

were stored at 4°C until analyzed. Slides for 1-h-old embryos were prepared following the

technique of Guo and Allen (1997) and slides for 6-h-old embryos were prepared following the

air drying technique of Thiriot-Quiéveux and Ayraud (1982). Chromosome counts were made

directly by microscope observation (Nikon Eclipse 50i with camera image acquisition

incorporated Nikon DS-Fi1) on apparently intact metaphases.

For 2-4 cell embryos, chromosome counts were made on at least 20 embryos. Each count

represented the contribution of chromosomes from the sire and the dam. For 6-h-old embryos, at

least 30 intact metaphases per cross were counted. A sample size of 30 is the minimal statistical

number typically accepted in cytogenetic studies (Leitão et al. 2001). In the case of 6-h-old

embryos, chromosome counts represent a random sample of cells from the population of

disaggregated embryos and cannot be attributed to any particular embryo. Aneuploidy incidence

was estimated as the total number of aneuploid metaphases divided by the total number of

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metaphases counted per cross. Average chromosome number from crosses using mosaic or non-

mosaic tetraploids was compared with a one-way analysis of variance (ANOVA parametric test)

at α=0.05.

Remaining embryos were reared in 40 liter tanks for 48h, at which time about 3,000 larvae

were examined for ploidy via FCM (Chaiton and Allen 1985). Relative DNA content and CV

were measured for the population of cells from each cross.

Results

Tetraploid broodstock

Males

Relative DNA content of gametic cells (di-haploid sperm) from mosaic and non-mosaic

males was the same, both with an average relative DNA content of 2.04 (Table 1). Di-haploid

sperm of mosaics had 0.52x the relative DNA content of gill tissue of tetraploids; for non-

mosaics 0.51x.

For tetraploid cell populations, average relative DNA content for somatic (gill) tissue of non-

mosaics was slightly higher than mosaics (3.99 vs. 3.92) (Table 1). Mosaic male oysters had two

populations of cells — tetraploid and “triploid.” Average relative DNA content of “triploid” cell

populations for mosaic males was 2.98 (n = 3) with an average CV of 3.50 (n = 3) (Table 1). On

average, the ratio of the mean relative DNA content of the “triploid” cell population to the mean

relative DNA content of the tetraploid population was 0.76 (n = 3), slightly higher than the

expected 0.75 (Table 1).

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Females

For tetraploid cell populations, average relative DNA content for somatic tissue (gill) of non-

mosaics was slightly lower than mosaics (3.95 vs. 4.00) (Table 1). All mosaic females had two

cell populations — tetraploid and “triploid”. Average relative DNA content of “triploid” cell

populations for mosaic females was 3.10 (n = 3) with an average CV of 3.59 (n = 3) (Table 1).

The ratio of the mean relative DNA content in the “triploid” cell population to the mean relative

DNA content of the tetraploid cell population was 0.78 (n = 3), also higher than the expected

0.75 ratio and slightly higher than the value in the males. The average percent of tetraploid cells

in females was 78.0 (n=6) (Table 1).

Larvae from diploid x tetraploid crosses

Day 2 — relative DNA content

FCM analysis of two-day-old larvae confirmed that all crosses produced 100% triploid

progeny. For larvae from male tetraploids, average relative DNA content was 2.95 (n = 6) and

average CV – 5.16 (n = 6). For larvae from female tetraploids, average relative DNA content

was 2.85 (n = 5) and average CV – 5.18 (n = 5) (Table 2). Relative DNA content of larvae from

males was significantly higher than that of tetraploid females (p=0.007). For comparisons of

relative DNA content between larvae from non-mosaic versus mosaic parents, there was no

difference, either for males (p=0.95) or females (p=0.51). Similarly, there were no significant

differences in CV between crosses made with non-mosaic or mosaic males (p=0.51) and non-

mosaic and mosaic females (p=0.92). The relative DNA content of larvae produced from the

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diploid female tester and random diploid male was 1.99 and CV – 5.50. For the larvae from the

diploid male tester and random diploid female the relative DNA content was 1.86 and CV – 5.65.

Cytogenetic analysis (6-h-old embryos)

Chromosome counts of 6-h-old triploid embryos from both female and male tetraploids, as

well as the diploid control, were analyzed (Figure 1, a-c). Cells from triploid embryos from both

non-mosaic and mosaic, males and females displayed a wide variation of chromosome number,

ranging from 16 to 38 chromosomes (Figure 2a, b). Despite this wide variation, all triploid

crosses had a modal chromosome number of 30 and the diploid – 20 (Table 3). More than half

of all metaphase spreads from triploid embryos were aneuploid. For male tetraploids, 64% of the

non-mosaic and 63% of the mosaic cells from the progeny were aneuploid. Additionally, 3%

and 6% of the cells from non-mosaic and mosaic progeny were diploid, respectively (Table 3).

For female tetraploids, 63% for the non-mosaic and 58% for the mosaic cells from the progeny

were aneuploid. Five per cent of the cells were diploid (n=20) for both non-mosaic and mosaic

progeny from female tetraploids (Table 3). There were no significant differences between the

proportion of aneuploids from male or female tetraploids (p=0.63), and no significant differences

in the proportion of aneuploids between progeny of non-mosaic and mosaic females (p=0.95), or

progeny of non-mosaic and mosaic males (p=0.72). Aneuploidy in the diploid controls was also

high – 36% and 53%. Of the aneuploid cells, one control cross had only hypoploid cells and the

second - 23% hypo- and 13% hyperploid.

Cytogenetic analysis (2-4 cell embryos)

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In order to better understand the origin of the high levels of aneuploidy observed in the 6-h-

old triploid embryos, we performed a cytogenetic analysis in the 2-4 cell triploid embryos (1-h-

old) from the same crosses for both female and male tetraploids and the diploid control. Only

the embryos from female tetraploids showed adequate metaphase spreads to perform

chromosome counts, perhaps owing to the physical nature of the eggs (Figure 1d). The

metaphase spreads of embryos resulting from diploid eggs (for both male tetraploids and the

diploid control) presented distended chromatin instead of the condensed chromosomes typical

from this phase, making the identification of the individual chromosomes impossible.

Hypoploid cells of 3n: 20, 26, 28 or 29 were observed in the embryos from both non-mosaic

and mosaic females (Figure 2b). Both embryos from non-mosaic and mosaics showed a modal

and an average chromosome number of 30. Compared with the 6-h-old triploid embryos, an

average of 8% of 2-4 cell embryos from both non-mosaic and mosaic parents were aneuploid

(Table 4). There was no significant difference observed between the progeny from mosaic and

non-mosaic tetraploid females (p=0.55).

Discussion

At the heart of this study was the question about the chromosome stability of tetraploid C.

virginica parents that have undergone the process of reversion to a state of becoming heteroploid

mosaics. Specifically, is the chromosome instability (CIN) of mosaic parents heritable? This

question was initially addressed by Matt and Allen (2014) using FCM, and peripherally touched

upon in cytogenetic studies of meiotic chromosomes by (Zhang et al. 2010b; Zhang et al. 2014).

In a recent paper by Benabdelmouna and Ledu (2015), heritability of CIN was further implicated

by observations from tetraploids derived through a completely different method of induction, the

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so-called direct method – produced by inhibiting the first polar body in a 2n x 2n cross (also see

below). Below, we discuss the observations of mosaicism from the parents in this study, our

observations of CIN in the crosses made from these parents, and insights that these observations

and other work tell us about the heritability of CIN in mosaic tetraploid oysters.

Non-mosaic and mosaic parents

The tendency for polyploid oysters to lose chromosomes over time has become an important

question for commercial production (e.g., Guo and Allen 1994a; Wang et al. 1999; Zhang et al.

2010a; Zhang et al. 2013; Matt and Allen 2014) since it was first reported by Allen et al. (1996)

20 years ago, as well as an interesting and possibly unique mechanism of chromosome loss

(Zhang et al. 2010a; Zhang et al. 2013). From the practical side, the short term consequences of

chromosome loss may affect commercial production in tetraploid x diploid hatchery output. The

longer term consequences concern how chromosome loss may affect the integrity of tetraploid

lines.

For tetraploid male and female parents, relative DNA content of tetraploid cells from non-

mosaics and mosaics were the same, confirming the observations in Matt and Allen (2014) and

reinforcing the idea that reversion occurs subsequent to the initial tetraploid condition.

“Triploid” cell populations, on the other hand, were not uniform. “Triploid” cells in male

mosaics were slightly hypo-triploid and “triploid” cells in female mosaics were slightly hyper-

triploid (Table 1). It is not difficult to envision why the “triploid,” i.e., the chromosomal

condition resulting from CIN, is variable and likely aneuploid, given the mechanism of

chromosome loss proposed by Zhang et al. (2010b) and Zhang et al. (2014). The so-called

clumping that sheds multiple chromosomes (and likely random numbers of them) over the course

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of reversion would be expected to yield imperfect levels of triploid (and diploid, if reversion

continued).

For gametic cells, i.e., di-haploid sperm of tetraploids, we found no difference in relative

DNA content or in its variance (CV) between the non-mosaics and mosaics by FCM. Similar

results were observed by Matt and Allen (2014) between non-mosaic and mosaic individuals of

C. virginica that underwent severe reversion or between non-mosaic and mosaic individuals of

C. gigas (McCombie et al. 2005a). That is, we could not detect aberrant meiotic products from

tetraploids that had, also, triploid somatic cells present. These results are consonant with

cytogenetic evaluations of chromosome pairing in meiosis of both triploid and tetraploid C. gigas

(Zhang et al. 2010b). In a similar analysis of spermatocytes from tetraploid C. gigas, one

tetraploid/ triploid mosaic was examined and only 1 of 47 spermatocytes (2%) was a triploid

(Zhang et al. 2014). Also in this same Zhang paper, the frequency of aneuploidy spermatocytes

was the same between the non-mosaic tetraploids and the one mosaic examined, suggesting that

aneuploidy in spermatocytes may be a function of being polyploid, but not necessarily to being a

heteroploid mosaic.

Crosses from non-mosaic and mosaic parents

Integrity of the gametes was also confirmed through the crosses that we tested. According

to FCM results, all the crosses performed in this study resulted in 100% triploid progeny even

when tetraploid mosaics were used as broodstock; no differences in relative DNA content were

evident between larvae from non-mosaic and mosaic parents. Similar results were observed in

previous studies (Guo et al. 1996; Matt and Allen 2014), confirming the consistency of this

method to produce triploid C. virginica larvae. There is no evidence that the mechanism giving

rise to mosaics affects DNA content of gametes in tetraploid parents. In fact, there was more of

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a difference in relative DNA content of triploid progeny between males and females than there

was between non-mosaics and mosaics (Table 2). Similarly, there was a noticeable difference in

the relative DNA content of diploid progeny produced from the female diploid tester to the

diploids produced from the male diploid tester. In contrast, Matt and Allen (2014) found DNA

content to be the same in 2-day-old triploid larvae produced from males and females.

In order to better understand the relationship between chromosome loss in tetraploid parents

and triploid progeny, chromosome counts of 6-h-old triploid embryos from both female and male

tetraploids (non-mosaic and mosaic), as well as a diploid control, were analyzed. One must bear

in mind that the aneuploidy we observed was from a population of cells comprised of

disaggregated embryos. Therefore, we are observing the pool of aneuploidy cells among many

individuals. High levels of aneuploidy, mainly hypotriploidy (58-64%, Table 3), prevailed in the

metaphases observed in these 6-h-old triploid embryos. At the same time, there was also a

relatively high level of aneuploidy in the two diploid controls (37%, 53%). In adult diploid

bivalves, aneuploidy was as high as 26% for C. gigas (Leitão et al. 2001) and 19% - 79% in adult

European clam, Ruditapes decussatus (de Sousa et al. 2011). In triploids of C. gigas aneuploidy

ranged from 6% - 12% in diploids and up to 56% in triploids (Guo and Allen 1997). Zhang et al.

(2010a) observed high levels of aneuploidy in triploid mosaic Pacific oysters (43% for induced

and 42% for mated [4n x 2n]) and Zhang et al. (2013) observed about the same level of

aneuploidy (46%) in chemically induced (i.e., G1 tetraploids from 3n x 2n mating) tetraploids.

Figure 3 is a graphic representation of data from Table 1 of Zhang et al. (2010a) showing the

chromosome distribution in adult triploids. It differs from those that we saw in this study by the

relative lack of chromosome counts in the mid-range, from 22-28. The difference in

chromosome distribution could be due to the fact that in the present work we analyzed triploid

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embryos, versus gill tissues of adult triploids in previous work, suggesting that highly aneuploidy

cells are lost over time and that chromosome numbers tend to stabilize, with time, around the

euploid state.

The patterns of aneuploidy from non-mosaic parents was the same as that for mosaic ones,

suggesting a lack of heritability for CIN, at least the type of CIN that leads to reversion (see

Chromosome instability and its consequences). The cytogenetic results from 6-h-old embryos

stands in contrast to those obtained from FCM on 2-day-old larvae. At 2-days old, there was

little to no sign of high levels of aneuploidy. Indeed, from the many FCM analyses of triploids

we have done in our lab, we had never had occasion to suspect aneuploidy in early development:

ploidy confirmation of triploid larvae for commercial purposes is always on larvae that are ≥ 2-

days old. We believe this lack of correspondence between aneuploidy at 6-h and 2 days old is

due to mortality of embryonic cells with severe aneuploidy, acting as a natural control on the

number of aneuploid cells produced by mosaics and non-mosaics alike.

Due to the high level of aneuploidy we observed in the 6-h-old triploid embryos, we decided

to further investigate the source of this phenomenon by performing additional chromosome

counts in 2-4 cell embryos (around 1-h-old embryos) from the same crosses. Again, non-

statistically significant differences were observed for chromosome loss or gain in 2-4 cell triploid

embryos produced from mosaic and non-mosaic. However, the levels of aneuploidy were

significantly lower in 2-4 cell embryos compared to the 6-h-old triploid embryos, with most of

the metaphases presenting 30 chromosomes in the former. Due to the increase of aneuploidy in

these first hours of early development, we can assume that the CIN that leads to the high levels

of aneuploidy in embryos occurs as a mitotic error during cell division. In polyploids across

taxa, aneuploidy, due to aberrant mitosis and errors in chromosomal segregation, is frequent and

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occurs to a much greater extent than in diploids (Comai 2005; Storchova and Kuffer 2008).

These errors during mitosis leading to abnormal chromosome numbers can culminate in the

activation of the apoptotic default pathway and cellular death (Castedo et al. 2004).

In our particular case, the 6h chromosomes counts could only be ascribed to individual cells,

not to individual embryos, as with the 2-4 cell data. Therefore, it was impossible to know

whether the distribution of chromosome counts represented a high occurrence of aneuploidy in a

small percentage of embryos or a lower occurrence of aneuploidy in the majority of embryos.

Judging from the tendency for larval cultures of triploids oysters to have similar survival to

diploids (Guo et al. 1996; Guo et al. 2009), it would seem that the loss of aneuploid cells may not

correspond to the loss of larvae, favoring the view that our cell population at 6h post-fertilization

represents a generally low level of aneuploidy among the entire population of embryos, which

also assumes low levels of aneuploidy are tolerated.

Chromosome instability and its consequences

We have been acutely interested in the consequences of CIN in tetraploids. It seems, based

on this and other work, that there is little evidence for increased levels of aneuploidy in progeny

as a consequence of reversion, i.e., mosaic tetraploids seem to produce predominantly euploid

gametes (Zhang et al. 2010a, Zhang et al. 2014, Matt and Allen 2014, this study). The

production of euploid gametes, or not, in mosaics is likely a completely different issue than the

mechanism(s) of chromosome loss in polyploids. That is, gamete production is a meiotic process

whereas chromosome loss yielding reversion (mosaics) seems to be a mitotic one.

Zhang et al. (2010a) and Zhang et al. (2013) proposed a mechanism of chromosome loss in

triploid C. gigas and Crassostrea ariakensis as well as in tetraploid C. gigas called “chromosome

clumping.” They observed that individuals with more chromosome clumps in their cells tended

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to have higher percentages of aneuploidy. Furthermore, the clumping was observed in mosaic

oysters. Whether clumping is the mechanism of reversion per se or the phenotypic manifestation

of the mechanism (discussed below) is open to interpretation, but the phenomenon of quantum

losses in chromosome number makes sense from all we have observed about mosaics.

Supporting evidence for losing chromosomes in “clumps” comes from several observations: with

FCM data, there is seldom evidence for intermediate ploidy types between the original and the

next level down in adult polyploids (e.g., between tetraploid and triploid); reversion happens

relatively quickly and begins at a relatively early age (Ritter and Allen 2015), which could

indicate sudden, quantum losses rather than gradual chromosome by chromosome loss; and, once

reversion commences, it is progressive over time (Allen unpublished data, Zhou 2002, Erskine

2003). To the latter point, in studies of polyploid yeast, if one chromosome is lost, additional

chromosomes are lost at increasingly higher frequencies (Mayer and Aguilere 1990).

Chromosome clumping may only be the phenotype of the actual mechanism for reversion.

The causative mechanism may be supernumerary centrosomes causing multipolar spindles

yielding chromosome clumps. Centrosomes coordinate important micro-tubule related

functions, including chromosome segregation and cytokinesis. Extra copies can result in

multipolar spindles and mitotic failures (Nigg 2002). Tetraploidy can result in supernumerary

centrosomes and, as studied mostly in mammalian systems, represents an important intermediate

on the route to aneuploidy by initiating chromosomal instability (Storchova and Kuffer 2008).

Tetraploid induction, as is performed in oysters, interferes with the normal cell cycle

development and may give rise to extra copies of centrosomes. Indeed, there are a number of

mechanisms posited for centrosome amplification, some associated with cytokinesis failure

leading to tetraploidy (Meraldi et al. 2002). It is not hard to imagine a multipolar spindle giving

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rise to a “clump” of chromosome that would result in a quantum loss of chromosomes from a

polyploid cell, a step on the way to reversion to the next ploidy down. Furthermore, the

hypothesis that mosaic formation and/ or aneuploidy is, at least partially, generated by

supernumerary centrosomes is widely supported in the literature (Storchova and Kuffer 2008).

It is interesting that besides the observation of chromosome clumping in tetraploid C. gigas,

Zhang et al. (2013) also observed asynchronous chromatic condensation that they said “could

account for losses in later divisions”. Duplication of centromeres occurs only once in each S

phase of the cell cycle but increasing the duration of S phase can lead to centrosome

amplification. The “later” chromosome losses suggested by Zhang et al. (2013) could be related

to delayed mitoses that allow abnormal centrosome duplication.

There is much to confirm about the role of supernumerary centrosomes as a process

accounting for mosaicism in polyploid oysters, so for now, it remains a hypothesis. At least it

seems consonant with observations of chromosome clumping. Such a mechanism might

reasonably be evoked for all types of polyploid shellfish, oysters being the prime example and

the only example for which mosaics have been documented so far. Observations of chromosome

clumping in mosaics were made on chemically induced triploids and mated (4n x 2n) triploids

(Zhang et al. 2010a) and chemically induced tetraploids (Zhang et al. 2013). Other evidence for

mosaics have been made through FCM, including chemical triploid C. gigas (Allen et al. 1996),

chemical triploid C. ariakensis (Zhou 2002), mated triploid C. ariakensis (Erskine 2003), mated

tetraploid C. virginica (Matt and Allen 2014) and, now, on direct tetraploids of C. gigas

(Benabdelmouna and Ledu 2015). It would seem, then, that the general condition of polyploidy

provides the substrate for subsequent chromosome loss.

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It is curious that there seems such a difference in the incidence of mosaicism in the several

types of tetraploids documented by Benabdelmouna and Ledu (2015). They compared three

types of tetraploids, which we will name after the authors responsible: GA – those induced from

triploid eggs (Guo and Allen 1994a); MC – those produced by fertilizing diploid eggs with

tetraploid sperm, followed by second polar body inhibition (McCombie et al. 2005b); and, BL –

those created directly by inhibiting the first polar body in a 2n x 2n cross (Guo et al. 1992a;

Benabdelmouna and Ledu 2015). In comparison of the three types of tetraploids at two years

old, the proportion of mosaics was GA – 45%, MC – 25%, and BL – 7%. These same results

were later confirmed in another year class: GA – 50% and BL – 5%. It is remarkable then that

there could be such a large difference between tetraploid oysters produced in three different

ways, especially when two of those (GA and BL) are produced by a similar cytological

manipulation.

The cytological manipulation that gave rise to the BL type oysters was first documented by

Guo et al. (1992a) and resulted in tetraploid embryos that did not survive through the larval

phase. More than two decades later, Benabdelmouna and Ledu (2015) were able to achieve the

zootechnical skill to keep these tetraploids alive. The chromosomal segregations that gave rise

to tetraploids when the first polar body of the diploid egg was inhibited were complicated but

clearly documented by Guo et al. (1992b). In summary, the segregations that produce the

tetraploid embryos were called either tripolar or separated bipolar segregations, either one of

which has to be the result of supernumerary centrosomes, by definition. GA tetraploids are also

produced by inhibiting the first polar body, but from triploid eggs, not diploid ones. The

chromosome segregation that gives rise to tetraploids under those circumstances – also

documented by Guo et al. (1992a, albeit in diploid eggs) – was called united bipolar segregation,

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and curiously does not involve supernumerary centrosomes. So what could be the explanation

for such a vast difference in reversion between GA and BL type oysters?

Benabdelmouna and Ledu (2015) proposed that the “manipulation of meiosis, particularly via

the blocking of the expulsion of the first polar body, has a much higher impact on triploid

oocytes than on diploid oocytes, due to the greater number of chromosomes that are concerned

by the meiotic segregation in triploid oocytes.” They suggested one of these impacts is initial

aneuploidy that accompanies the inhibition of polar body I. Another proposed impact was a

genetic predisposition transmitted by the triploid female. As to the first “impact,” it should be

noted that the cytogenetic divisions giving rise to GA tetraploids are inherently more “normal” (a

bipolar segregation) that those giving rise to BL tetraploids (a tripolar segregation or a

quadripolar segregation). So it is possible that the level of aneuploidy is the same after either

GA or BL tetraploid induction, and, arguably, GA tetraploids might be less aneuploid because of

the lack of a multipolar spindle to give rise to tetraploids. To verify any difference in initial

levels of aneuploidy between GA and BL, chromosome counts in early embryos will be needed

in future studies.

The second “impact,” that of genetic predisposition for reversion, which Benabdelmouna

and Ledu (2015) hypothesized would be “directly and fully transmitted by triploid oocytes” is

supported by the fact that MC tetraploids (which had ½ of their genetic material inherited from

the genetically predisposed tetraploid parent) had intermediate levels of reversion between GA

and BL tetraploids. The idea that CIN is heritable is intriguing, although counter to the argument

that CIN is an inherent feature of polyploidy in the first place (Comai 2005; Storchova and

Kuffer 2008). Whatever this genetic effect is, it seems to be additive (cf. Benabdelmouna and

Ledu 2015). However, in this study and that of Matt and Allen (2014), we found no evidence of

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heritability of CIN between non-mosaic and mosaic parents, as measured through both FCM and

chromosome counts in triploid C. virginica. It is possible that somatic chromosome loss is of

less concern for the long-term chromosomal integrity of 4n x 4n crosses than would be suggested

by the levels of reversion observed in tetraploid adults.

Acknowledgements

We would like to thank the entire staff of the Aquaculture Genetics and Breeding Technology

Center (ABC) for their technical help, without whose expertise this research would not be

possible. Special thanks to Kate Ritter for her assistance with flow cytometry, Shelley Katsuki

for her assistance with breeding and rearing the oysters and Eric Guévélou for translating the

abstract into French. This work was partly funded through the National Science Foundation’s

Research Experience for Undergraduates (REU) Program (grant # NSF OCE 1062882). This

paper is Contribution No. XXXX of the Virginia Institute of Marine Science, College of William & Mary.

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Tables

Table 1. Parents: Flow cytometric analysis of males and females, mosaics and non-mosaics of C.

virginica broodstock used for crosses showing relative DNA content (Rel DNA) and coefficient

of variation (CV) for cell populations of sperm (2C) and somatic cells: tetraploid (4n) and

triploid cells (3n). For mosaics, the ratio of triploid to tetraploid relative DNA content (3n/4n

ratio) and percentage of tetraploid cells (% 4n) for each individual is shown. NA – samples not

available.

SPERM SOMATIC ANALYSIS

2C Rel

3n Rel

4n Rel

3n/4n

DNA CV DNA CV

DNA CV

Ratio % 4n

MALES

NM 1 NA NA -- -- 4.03 3.24 -- 100

NM 2 2.03 3.05 -- -- 3.96 3.25 -- 100

NM 3 2.05 3.73 -- -- 3.99 3.25 -- 100

NM mean 2.04 3.39 3.99 3.25

M 1 2.97 3.97 3.95 3.57 0.75 66 M 2 2.04 3.39 3.06 3.06 3.92 3.24 0.78 42 M 3 2.04 3.58 2.92 3.46 3.90 3.25 0.75 60

M Mean 2.04 3.49 2.98 3.50 3.92 3.35 0.76 56.0

Mean all 2.04 3.44

2.98 3.50

3.96 3.30

0.76 --

SD all 0.01 0.29

0.07 0.46

0.05 0.13

0.02 --

FEMALES NM 1 -- -- -- -- 4.01 3.64 -- 100

NM 2 -- -- -- -- 3.89 3.38 -- 100 MN 3 -- -- -- -- 3.95 3.16 -- 100

NM mean 3.95 3.39

M 1 -- -- 3.11 3.95 4.03 3.41 0.77 50

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M 2 -- -- 3.10 3.41 3.99 3.35 0.78 62 M 3 -- -- 3.09 3.41 3.97 4.21 0.78 56

M mean 3.10 3.59 4.00 3.66 0.78 56.0

Mean all -- -- 3.10 3.59 3.97 3.53 0.78 --

SD all -- -- 0.01 0.31 0.05 0.37 0.01 --

Table 2. Progeny: Flow cytometric analysis of triploid C. virginica larvae from day 2 showing

relative DNA content (Rel DNA) and coefficient of variation (CV). Larvae were produced by

crossing either non-mosaic (NM) or mosaic (M) tetraploid males × a diploid female tester or

non-mosaic or mosaic tetraploid females × diploid male tester. The female tester diploid was

crossed to a random diploid male; the male tester diploid was crossed to a random diploid

female. NA – samples not available.

Rel

DNA CV

MALES

NM1 2.97 4.75

NM2 2.90 5.51

NM3 2.98 5.07

M1 2.95 5.21 M2 2.90 5.51 M3 3.01 4.89

Mean 2.95 5.16

SD 0.04 0.32

Control 1.99 5.50

FEMALES

NM1 2.84 4.47 NM2 2.86 4.91 NM3 2.80 5.45

M1 2.89 6.01 M2 NA NA M3 2.85 5.07

Mean 2.85 5.18

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SD 0.03 0.58

Control 1.86 5.65

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Table 3. Chromosome count data and percentage of spreads among different ploidy classifications for 6-h-old triploid embryos of C.

virginica produced by crossing non-mosaic (NM) or mosaic (M) tetraploid males × a diploid female tester or non-mosaic or mosaic

tetraploid females × diploid male tester. n = number of metaphase spreads.

NON-MOSAIC MOSAIC

Control NM1 NM2 NM3 M1 M2 M3

MALES

NO. n 30 36 31 30 32 33 31 38 34

mode 20 30 30 30 30 30 30 30 30

mean 20 27.1 27.1 27.3 27.2 26.2 27.0 27.7 27.0

PERCENT

triploid 0 33 23 43 33 24 29 39 31

diploid 63 6 0 3 3 9 6 3 6

hypoploid 23 58 71 50 60 67 65 58 63

hyperploid 13 3 6 3 4 0 0 0 0

FEMALES NO. n 36 30 32 30 31 34 30 31 32

mode 20 30 30 30 30 30 30 30 30

mean 19 26.3 26.2 28.1 27.0 26.0 27.3 27.6 27.0

PERCENT triploid 23 31 40 32 26 37 45 36

diploid 47 10 6 0 5 3 10 3 5

hypoploid 53 63 62 50 59 68 53 48 56

hyperploid 0 3 0 10 4 3 0 3 2

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Table 4. Chromosome count data and percentage of spreads among different ploidy

classifications for 2-4 cell triploid embryos of C. virginica produced by crossing non-mosaic

(NM) or mosaic (M) tetraploid females × a diploid male tester.

NON-MOSAIC MOSAIC

NM1 NM2 M1 M2 M3

NO. n 27 21 24 28 23 24 25

mode 30 30 30 30 30 30 30 mean 29.3 29.9 29.6 29.9 29.8 30.0 29.9

PERCENT triploid 85 95 90 93 87 96 92

diploid 4 0 2 0 0 0 0

hypoploid 11 5 8 7 13 4 8 hyperploid 0 0 0 0 0 0 0

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Figure captions

Figure 1. Examples of metaphase chromosomes from C. virginica embryos. (a) Diploid 2n =

20 from 2n control. (b) Hypoploid 3n = 29 from 6-h-old embryo from 4n male non-mosaic x 2n

female. (c) Eutriploid 3n = 30 from 6-h-old embryo from 4n male mosaic x 2n female. (d)

Eutriploid 3n = 30 from 2 cell embryo from 4n female mosaic x 2n male. Scale bar=5µm.

Figure 2a. Male tetraploid parents: Frequency distribution of chromosome number of cells

from 6-h-old triploid embryos of C. virginica produced by crossing either tetraploid non-mosaic

or mosaic tetraploid males with a diploid tester female and the diploid control.

Figure 2b. Female tetraploid parents: Frequency distribution of chromosome number of cells

from for 6-h-old triploid embryos and for 2-4 cell stage embryos of C. virginica produced by

crossing either tetraploid non-mosaic mosaic tetraploid females with a diploid tester male and

the diploid control.

Figure 3. Frequency distribution of chromosome number for adult, triploid, mosaic C. gigas,

from Table 1 (Zhang et al. 2010a). Tetraploids were G1 obtained from chemical induction

method of Guo and Allen (1994).

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Figure 1. Examples of metaphase chromosomes from C. virginica embryos. (a) Diploid 2n = 20 from 2n control. (b) Hypoploid 3n = 29 from 6-h-old embryo from 4n male non-mosaic x 2n female. (c) Eutriploid 3n = 30 from 6-h-old embryo from 4n male mosaic x 2n female. (d) Eutriploid 3n = 30 from 2 cell embryo

from 4n female mosaic x 2n male. Scale bar=5µm. 104x104mm (300 x 300 DPI)

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Figure 2a. Male tetraploid parents: Frequency distribution of chromosome number of cells from 6-h-old triploid embryos of C. virginica produced by crossing either tetraploid non-mosaic or mosaic tetraploid males

with a diploid tester female and the diploid control.

152x221mm (300 x 300 DPI)

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Figure 2b. Female tetraploid parents: Frequency distribution of chromosome number of cells from for 6-h-old triploid embryos and for 2-4 cell stage embryos of C. virginica produced by crossing either tetraploid

non-mosaic mosaic tetraploid females with a diploid tester male and the diploid control.

155x227mm (300 x 300 DPI)

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Figure 3. Frequency distribution of chromosome number for adult, triploid, mosaic C. gigas, from Table 1 (Zhang et al. 2010a). Tetraploids were G1 obtained from chemical induction method of Guo and Allen

(1994). 168x105mm (300 x 300 DPI)

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